Supplementary Material for: Increased Calcification in Osteoprotegerin-Deficient Smooth Muscle Cells: Dependence on Receptor Activator of NF-κB Ligand and Interleukin 6 Callegari A. Coons M.L. Ricks J.L. Rosenfeld M.E. Scatena M. 10.6084/m9.figshare.5126251.v1 https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Increased_Calcification_in_Osteoprotegerin-Deficient_Smooth_Muscle_Cells_Dependence_on_Receptor_Activator_of_NF-_B_Ligand_and_Interleukin_6/5126251 <b><i>Objective:</i></b> Vascular calcification is highly correlated with cardiovascular disease morbidity and mortality. Osteoprotegerin (OPG) is a secreted decoy receptor for receptor activator of NF-κB ligand (RANKL). Inactivation of OPG in apolipoprotein E-deficient (ApoE-/-) mice increases lesion size and calcification. The mechanism(s) by which OPG is atheroprotective and anticalcific have not been entirely determined. We investigated whether OPG-deficient vascular smooth muscle cells (VSMCs) are more susceptible to mineralization and whether RANKL mediates this process. <b><i>Results:</i></b> Lesion-free aortas from 12-week-old ApoE-/-OPG-/- mice had spotty calcification, an appearance of osteochondrogenic factors and a decrease of smooth muscle markers when compared to ApoE-/-OPG+/+ aortas. In osteogenic conditions, VSMCs isolated from ApoE-/-OPG-/- (KO-VSMC) mice deposited more calcium than VSMCs isolated from ApoE-/-OPG+/+ (WT-VSMC) mice. Gene expression and biochemical analysis indicated accelerated osteochondrogenic differentiation. Ablation of RANKL signaling in KO-VSMCs rescued the accelerated calcification. While WT-VSMCs did not respond to RANKL treatment, KO-VSMCs responded with enhanced calcification and the upregulation of osteochondrogenic genes. RANKL strongly induced interleukin 6 (IL-6), which partially mediated RANKL-dependent calcification and gene expression in KO-VSMCs. <b><i>Conclusions:</i></b> OPG inhibits vascular calcification by regulating the procalcific effects of RANKL on VSMCs and is thus a possible target for therapeutic intervention. 2014-03-07 00:00:00 Osteoprotegerin Receptor activator of NF-kappaB ligand Interleukin 6 Smooth muscle cells Vascular calcification Osteochondrogenic differentiation